Coronaviruses (CVs) are widespread, medically important respiratory and enteric pathogens of humans and many domestic animals, causing a significant portion of human upper respiratory infections. These enveloped viruses contain a positive(+)-sense, single-stranded RNA genome that is the largest (approximately 30 kb) of all the RNA viruses. Many questions remain to be answered about the molecular details of assembly of enveloped RNA viruses. The studies proposed herein focus on the mechanism of assembly of mouse hepatitis virus (MHV) that acquire their envelopes by a nucleocapsid independent manner at membranes of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). Virus-like-particles (VLPs) assemble when only the small envelope (E) and membrane (M) proteins are coexpressed. We hypothesize that E performs its role through its interplay with M and the ERGIC lipid membranes and possibly host proteins. Previous studies addressing the role of E and M in virus assembly relied on virus-infected cells, VLPs, and targeted RNA recombination. With the availability of a new MHV infectious clone we are uniquely positioned to directly manipulate the virus genome to study the mechanism by which E and M function in virion assembly. In Aim 1 we will focus on understanding the role of the E and M proteins in targeting assembly to intracellular membranes. We will precisely identify the site of intracellular localization for E. The retention signal in E and domains involved in potential co-localization with M will be identified. The role of E, M and host proteins will be studied to determine their roles in assembly/budding at intracellular membranes. Aims 2 and 3 are to understand the roles of the E and M proteins in virion formation using VLPs, the MHV infectious clone and replicons. We will use biochemical and microscopic analyses of chimerics and site-specific mutants of E and M expressed from various vectors and the MHV infectious clone to study the proteins, subcellular fractions and assembly of wild-type and altered VLPs and viruses. Information gained from this study can be used to (i) increase our understanding of fundamental mechanisms by which viruses of medical importance acquire their envelopes at intracellular membranes, (ii) identify major targets for antiviral drug development, (iii) assist in development of CV heterologous gene expression vectors for vaccine use, and (iv) contribute to understanding of protein-protein and protein-membrane interactions and transport.